Studies On Biological Characteristics And Establishment Of Donor Cells Line On Nuclear Transfer In Holstein Cows And Romilly·Hills

The objective of study is to establish the donor cells line on nuclear transfer of Holstein cows andRomilly·Hills, the characteristic of donor cell were also studied. The results as follows:1.In experiment 1, In vitro culture system of Holstein cows and Romney ram ear skin wasinvestigated in this study. The skin fibroblasts line was established. The method of tissue-sticking andnon-thoroughly cold enzyme digestion in primary culture were used. Cell could be derived by outgrowth ofmigrating cells from fragment of tissues. In subculture, fibroblast and epithelial could be purifiedaccording to their different sensibility to trypsin. Complex cultures treated with pro-cold TE at control time,the dispersed cells were mainly fibroblasts. The purified fibroblasts cells line could be obtained by 2~3passages. We also tested the growth curve of the cell line. In conclusion, this culture system was suitableto culture fibroblasts, the cell can be survived in DMEM, DMEM/F12 or RPMI1640 as basic mediacontaining 10% NCS,100IU/ml Penicillin,100IU/ml Streptomycin.2. In experiment 2, in vitro culture system of Holstein cows ovary granulosa cells and sheep ovarygranulosa cells was explored. The results showed that the system was suitable, and the population doubletime(PDT) is 1.14d, 1.08d respectively.3. In experiment 3,effects on the cell cycle by the different growth conditions of sheep ovarygranulosa cells, different the number of passage and different the methods of synchronization werestudied. Examined by FACS showed that the percentage of G0/G1 cell of the 3rd and the 13th passage were76.6±1.7%,83.4±1.65%,84.53±2.27%and66.8±1.84%,72.8±2.09%,76±3.41%at the different confluencerespectively. the percentage of G0/G1 of the 3rd passage cells undergoing lower-temperature 6 hours andserum starvation for 3 days were 93.3±2.1%,92.43±1.02%respectively. The results indicated that thepercentage of cells at G0/G1 phase were higher that that of higher passage(p<0.05), the methods oflower-temperature and serum starvation both could increase the percentage of cells at G0/G1 phaseextremely.